Comparison of hepatitis B viral loads and viral antigen levels in child-bearing age women with and without pregnancy

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1 Xu et al. BMC Pregnancy and Childbirth (2018) 18:292 htts://doi.org/ /s RESEARCH ARTICLE Oen Access Comarison of heatitis B viral loads and viral antigen levels in child-bearing age women with and without regnancy Chenyu Xu 1, Jingli Liu 2, Lanhua Liu 3, Yongchun Bi 2, Biyun Xu 4, Jie Chen 5, Biao Xu 3, Tingmei Chen 1, Yali Hu 5 and Yi-Hua Zhou 2,6* Abstract Background: Pregnancy is a unique hysiological condition with the cellular immune functions comromised at some extents to allow the mature of growing fetus. Whether regnancy may influence the relication of heatitis B virus (HBV) is less studied. The resent study aimed to investigate the influence of regnancy on the relication of HBV and exression of viral antigens by comaring the levels of HBV DNA and viral antigens in regnant and non-regnant women. Methods: A total of 727 HBsAg-ositive serum samles, collected from 214 regnant women and 513 non-regnant women of childbearing age, were included. Based on the regnancy status, subjects were divided into four grous: nulliarous (n = 158), regnant (n =214),7 12 months ostartum (n =170),and2 5 years ostartum (n = 185). The levels of heatitis B surface antigen (HBsAg) and heatitis B e antigen (HBeAg) were quantitatively measured with microarticle enzyme immunoassay. HBV DNA levels were detected by fluorescent real-time PCR. Results: The median ages of four grous were 25.0, 25.3, 26.2 and 29.3 years, resectively ( <0.01).HBeAg-ositive roortions were 34.2, 33.6, 35.3 and 29.2%, resectively ( = 0.624). HBV DNA levels in HBeAg-ositive women were higher than those in HBeAg-negative women (7.88 vs 2.62 log IU/ml, < 0.001). HBV DNA levels in the four grous with ositive HBeAg were 7.8, 7.7, 8.0 and 8.0 log IU/ml, resectively ( = 0.057), while HBsAg titers were 4.4, 4.5, 4.6 and 4.8 log IU/ml ( = 0.086) and HBeAg titers were 3.1, 3.0, 3.1 and 3.0 log S/CO ( = 0.198). In the four grous with negative HBeAg, HBV DNA levels were 2.3, 2.6, 2.5 and 2.8 log IU/ml, resectively ( = 0.085), while HBsAg titers were 3.1, 3.3, 3.3 and 3.0 log IU/ml ( =0.06). Conclusions: Serum levels of HBV DNA and viral antigens showed no significant changes in nulliarous, regnant, and ostartum women, regardless of the HBeAg status. The results indicate that regnancy has little influence on the relication of HBV and the exression of viral antigens. Keywords: Pregnancy, HBV relication, Exression of HBsAg and HBeAg * Corresondence: zgr03summer@126.com Chenyu Xu and Jingli Liu contributed equally to this work. 2 Deartments of Laboratory Medicine and Infectious Diseases, Nanjing Drum Tower Hosital and Jiangsu Key Laboratory for Molecular Medicine, Nanjing University Medical School, 321 Zhongshan Road, Nanjing , Jiangsu, China 6 Deartment of Infectious Diseases, Nanjing Drum Tower Hosital, Nanjing Medical University, Nanjing , Jiangsu, China Full list of author information is available at the end of the article The Author(s) Oen Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (htt://creativecommons.org/licenses/by/4.0/), which ermits unrestricted use, distribution, and reroduction in any medium, rovided you give aroriate credit to the original author(s) and the source, rovide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (htt://creativecommons.org/ublicdomain/zero/1.0/) alies to the data made available in this article, unless otherwise stated.

2 Xu et al. BMC Pregnancy and Childbirth (2018) 18:292 Page 2 of 7 Background Chronic heatitis B virus (HBV) infection is a serious health roblem because of the severe sequelae such as liver cirrhosis and cancer. It is estimated that there are aroximately 250 million chronic carriers worldwide [1]. Transmission of HBV from carrier mothers to their children is an imortant cause of chronic infection in heatitis B endemic areas. Before the availability of heatitis B immunoglobulin (HBIG) and heatitis B vaccine, 10 30% and 70 90% of the children born to HBV carrier mothers with negative heatitis B e antigen (HBeAg) and to HBeAg-ositive carrier mothers had been chronically infected with HBV resectively. Measures for reventing mother-to-child transmission of HBV, combined use of HBIG and heatitis B vaccine [2, 3], have been imlemented in most countries. The combined immunorohylaxis measures are highly effective; nearly zero and < 10% transmission occurred in children born to HBeAg-negative and HBeAg-ositive carrier mothers resectively [4 7]. More recently, antiviral theray in late regnancy in regnant women with high viral load is increasing [8, 9], although the safety of antiviral drugs remains further observations [10 12]. On the other hand, the influence of regnancy on the relication of HBV in regnant women infected with HBV is less studied. A survey in Massachusetts General Hosital, Boston, USA, showed a high frequency of inadequate care of mothers with chronic heatitis B ost-regnancy [13]. Pregnancy is a unique hysiological condition. A successful regnancy requires the tolerance of maternal immune system to the growing fetus as a fetus is actually a semi-allogeneic graft, in which half of the genomes are derived from the father. Pregnancy reresents a eriod of time with articular changes of immunological functions, comromised at some extents to allow the mature of a fetus [14]. Usually, the tolerance of immune resonse occurs in the cellular immune arm. Pregnant women are suscetible to reactivation and infection of latent viruses, such as heres zoster virus and cytomegalovirus [15, 16], or elevated relication level of infected virus, such as heatitis C virus [17]. It is generally considered that the relication of HBV is increased during regnancy due to the immunosuression [18], but relevant studies are limited and the conclusions are inconsistent [19, 20]. The resent study comared the levels of HBV DNA and viral antigens in regnant women with those in non-regnant women to clarify whether the relication of HBV is increased during regnancy. Methods Study subjects Totally 727 women who were ositive for heatitis B surface antigen (HBsAg) and had normal alanine aminotransferase levels were enrolled from October 2009 to March The median age was 26.0 ( ) years old. All women were negative for antibodies against heatitis C virus and human immunodeficiency virus. Those who had history of antiviral theray or were taking antiviral drug were excluded. Additionally, the liver function tests of these women were basically normal, in which both alanine transaminase and asartate transaminase were < 50 IU/L (1.25 uer normal limitation) and total bilirubin levels were < 21 μmol/l. Based on the regnancy and reroductive history, the women were divided into four grous: 158 women who never had history of regnancy in grou A (nulliara), 214 women who were in the third trimester (from gestation 28 weeks to just before delivery) in grou B (regnancy), 170 women at 7 12 months ostartum in grou C (uererant), and 185 women at 2 5 years ostartum in grou D (uererant). The eriheral blood samles were collected and serum samles were searated by centrifugation and frozen at 30 C. All samles were sent to Nanjing Drum Tower Hosital in ice box for the laboratory testing, including quantification of HBV DNA and viral antigen and genotying. This study, including the form of informed consent, was aroved by the ethics committee of each of the three articiating hosital, including Nanjing Drum Tower Hosital (aroval no ), Zhenjiang Fourth Peole s Hosital (aroval no ), and Taixing Peole s Hosital (aroval no ). The study was erformed in accordance with the ethical standards in the Declaration of Helsinki. Written informed consent was obtained from each woman. Detection of heatitis B serological markers and HBV DNA Serological markers of heatitis B, including heatitis B surface antigen (HBsAg), HBeAg, antibody against HBeAg (anti-hbe) and antibody against heatitis B core antigen (anti-hbc), were qualitatively detected with commercial enzyme-linked immunosorbent assay reagents (Shanghai Kehua Bio-Engineering Co. Ltd., Shanghai, China. Products numbers: HBsAg, YBS ; HBeAg, 20,163,400,144; anti-hbe, YZB/ ; anti-hbc 20,163,400,143). The measurements were erformed according to the manufacturer s instructions, with using 50 μl serum for detecting HBsAg, HBeAg, and anti-hbe resectively, and 50 μl 30-fold diluted serum (1.7 μl serum and 48.3 μl normal solution) for detecting anti-hbc. The levels of HBsAg and HBeAg were further quantitatively measured with microarticle enzyme immunoassay reagents (ARCHI- TECT, Abbott, North Chicago, USA); when the HBsAg levels were higher than the uer detection limit (250 IU/ml), the sera were diluted to 1:20 to 1:1000 to obtain a reading within the range of the calibration curve. All the measurements of quantification of

3 Xu et al. BMC Pregnancy and Childbirth (2018) 18:292 Page 3 of 7 HBsAg and HBeAg were comleted within one day with same batch reagents by an investigator (YB) who was unaware of the serum identity. Serum HBV DNA levels were quantitatively detected with real-time fluorescence quantitative PCR reagents (Shenyou Biotechnology, Shanghai, China; Products numbers, YZB/ ) on a thermal cycler (Alied Biosystems 7500 Real-time PCR System, Singaore), with the detection limit 100 IU/ml. To minimize the variation in measuring HBV DNA levels, all samles were measured with same batch reagents by the same investigator (JL) who was blinded to samle identity and the test was erformed strictly according to the manufacturer s instructions. HBV genotying The serum DNA was extracted from 200 μl serum with the roteinase K digestion and henol/chloroform extraction method and dissolved in 20 μl Tris-EDTA buffer as reviously reorted [21, 22]. The S gene was amlified with nested olymerase chain reaction (PCR) with the rimers C1 (nt , 5 -YTGGCCAAAA TTCGCAGTC-3 ) and C2 (nt , 5 -AAACCCCAR- RAGACCCACAA-3 ) for the first round amlification, and C3 (nt , 5 -CTCCARTCACTCACCAAC-3 and C4 (nt , 5 -TGACAKACYTTCCAATCAAT-3 ) forthe second amlification. The resultant PCR roducts were urified by PCR urification kit (Tiangen Biotech, Beijing, China), followed by thermocycling reaction using BigDye Terminator v3.1 sequencing reagents (ABI, USA), and then directly sequenced on an ABI 3130 Gene Analyzer. The S sequences were aligned with reference sequences that included genotyes A to H, which were obtained from the NCBI website (URL: htt:// genotying/view.cgi?db=2). HBV genotyes were determined by hylogenetic analysis based on the S gene sequences as reviously described [22]. Statistical analysis The statistical software SPSS 11.0 (SPPS Inc., Chicago, IL, USA) was used for the analysis. Inter-grou comarison was carried out after the quantitative levels of HBV DNA, HBsAg and HBeAg were converted to logarithmic values. Measurement data was descried with median and range, inter-grou differences were analyzed with Kruskal-Wallis test, and then Nemenyi ost-hoc test was used to analyze the difference between any two grous. Categorical data were described with rate and inter-grou differences were analyzed with χ 2 test or the Fisher exact test. A value < 0.05 with two-tailed test was considered statistically significant. Results Demograhic and virological characteristics in women The median age of women in each grou and roortion of women with ositive HBeAg were resented in Table 1. The age difference between nulliara and regnancy grous had no statistical significance, while the age in uererant 7 12 months ostartum and uererant 2 5 years ostartum grous was slightly higher than that in two other grous ( < 0.05). The HBeAg ositive roortion in the four grous was comarable, with the overall roortion of 33.0% (Table 1). The median serum HBV DNA level in women with ositive HBeAg was significantly higher than that in those with negative HBeAg (7.88 vs 2.62 log IU/ml, < 0.001). HBV genotyes were determined in 689 women and were not determined in 38 other women due to the undetectability Table 1 Demograhic and virological characteristics in 727 HBsAg-ositive regnant women and women with different reroductive histories Total (n = 727) Nulliara (n = 158) Pregnancy (n = 214) Puererant, 7 12 months ostartum (n = 170) Puererant, 2 5 years ostartum (n = 185) Age (year) 26.0 ( ) 25.0 ( ) 25.3 ( ) 26.2 ( ) a 29.3 ( ) a, b < 0.05 Positive HBeAg (%) 240 (33.0) 54 (34.2) 72 (33.6%) 60 (35.3) 54 (29.2) HBV DNA, log IU/ml (min-max) 3.1 ( ) 3.0 ( ) 3.1 ( ) 3.2 ( ) 3.1 ( ) HBsAg, log IU/ml (min-max) 3.7 ( ) 3.4 ( ) 3.7 ( ) 3.8 ( ) 3.5 ( ) Genotye c B (%) 303 (44.2) 59 (44.4) 70 (33.5) d 77 (46.7) 97 (54.2) C (%) 383 (55.8) 74 (55.6) 139 (66.5) d 88 (53.3) 82 (45.8) The units of numbers in the arentheses are indicated in the first column HBeAg heatitis B e antigen, HBsAg heatitis B surface antigen, HBV heatitis B virus a < 0.05 comared with the age of nulliaras and regnant women b < 0.05 comared with uererant women 7 12 months ostartum c HBV genotye was not determined in 38 women because of failure in the amlification of HBV DNA, including 24 nulliaras, 4 regnant women, 5 uererants 7 12 months ostartum and 5 uererants 2 5 years ostartum. Additionally, there were two women infected with genotye D and one woman infected with genotye G. Since the numbers for genotyes D and G were too low to be meaningfully comared, we did not include these three women in the analysis d = 0.043, 0.010, and < comared with the frequencies of genotyes B and C in nulliara grou, uererant 7 12 months ostartum and uererant 2 5 years ostartum resectively. The differences between any other grous had no statistical significance (all >0.05)

4 Xu et al. BMC Pregnancy and Childbirth (2018) 18:292 Page 4 of 7 Table 2 Comarison of virological characteristics in 240 HBeAg-ositive regnant women and women with different reroductive history Nulliara (n = 54) Pregnancy (n = 72) Puererant, 7 12 months ostartum (n = 60) Puererant, 2 5 years ostartum (n = 54) Age (year) 25.0 ( ) 23.8 ( ) 25.6 ( ) 28.9 ( ) a < 0.05 HBV DNA, log IU/ml (min-max) 7.8 ( ) 7.7 ( ) 8.0 ( ) 8.0 ( ) HBsAg, log IU/ml (min-max) 4.4 ( ) 4.5 ( ) 4.6 ( ) 4.8 ( ) HBeAg, log S/CO 3.1 ( ) 3.0 ( ) 3.1 ( ) 3.0 ( ) (min-max) Genotye b B (%) 18 (34.0) 24 (33.3) 25 (41.7) 16 (29.6) C (%) 35 (66.0) 48 (66.7) 35 (58.3) 38 (70.4) HBeAg heatitis B e antigen, HBsAg heatitis B surface antigen, HBV heatitis B virus a < 0.05 comared with the age of three other grous b HBV genotye was not determined in one woman in nulliara grou due to the undetectability of HBV DNA of HBV DNA (Table 1). The main genotyes were B and C. Overall, comared with the nulliarous and arturient women, the regnant women had no increased serum levels of HBV DNA and HBsAg (Table 1). Comarison of levels of HBV DNA and viral antigens in carrier women with ositive HBeAg The resence of HBeAg indicates the active relication of HBV. To rule out the otential influence of different roortions of HBeAg ositivity in the four grous of women on the viral relication, we searately comared the levels of HBV DNA and viral antigens in HBeAg-ositive and -negative women. As shown in Table 2, thevirological characteristics and the frequency of genotyes were similar among the four grous. The genotyes were exclusively B and C, which is in agreement with the findings in China [23]. There was no statistically significant difference in serum HBV DNA level between regnant women and non-regnant women or women after childbirth ( = 0.057). Similarly, serum HBsAg and HBeAg concentrations were each comarable among different grous resectively. To exclude the otential influence of extremely high or low HBV DNA levels in some women on the overall viral levels, we analyzed the frequency of high ( 6 log IU/ ml), moderate (3 < 6 log IU/ml) and low (< 3 log IU/ml) serum HBV DNA levels in the four grous of HBeAg ositive women (Table 3). The frequencies of high, moderate and low viral levels had no significant difference in the four grous. Comarison of HBV DNA and HBsAg levels in HBV carrier women with negative HBeAg Negativity of HBeAg, regardless of the ositivity of anti-hbe, indicates the low relication of HBV. We comared the levels of HBV DNA and HBsAg in the four grous of carrier women with negative HBeAg. Table 4 shows that the levels of HBV DNA in the regnant women were comarable with those in the nulliarous women, uererants 7 12 months and 2 5 years ostartum, resectively. The frequency of high ( 6 log IU/ml), moderate (3 < 6 log IU/ml) and low (< 3 log IU/ml) levels of HBV DNA in these four grous were comarable (Table 5). Similarly, the levels of HBsAg in the four grous of women had no significant difference (Table 4). Discussion The data in the resent study show that the levels of HBV DNA and viral antigens in the regnant women were comarable with those in nulliaras, uererants 7 12 months and 2 5 years ostartum, resectively, indicating that the relication of HBV and exression of viral antigens during regnancy are not increased in site of the resence of immune tolerance during regnancy. In individuals infected with HBV, the roortion of ositive HBeAg is decreased with age. In East Asia, the ositive HBeAg roortion in HBV carrier women aged years is % [24]. In the resent study, the age distribution in the articiant is relatively homogenous ( years old). Only the women 2 5 years ostartumhadtherelativelyhigherage,andtheroortionof Table 3 The frequency of high, moderate, and low level of HBV DNA in 240 carrier women with ositive HBeAg HBV DNA (log IU/ml) Nulliara (n = 54) Pregnancy (n = 72) Puererant, 7 12 months ostartum (n = 60) Puererant, 2 5 years ostartum (n = 54) < 3 (%) 3 (5.5) 2 (2.8) 3 (5.0) 1 (1.9) < 6 (%) 7 (13.0) 4 (5.5) 5 (8.3) 3 (5.5) (%) 44 (81.5) 66 (91.7) 52 (86.7) 50 (92.6) HBeAg, heatitis B e antigen; HBV, heatitis B virus. The units of numbers in the arentheses are indicated in the first column

5 Xu et al. BMC Pregnancy and Childbirth (2018) 18:292 Page 5 of 7 Table 4 Virological characteristics in HBeAg-negative carrier women in 487 regnant women and women with different regnant histories Nulliara (n = 104) Pregnancy (n = 142) 7 12 months ostartum (n = 110) 2 5 years ostartum (n = 131) Age (year) 25.0( ) 25.0 ( ) 27.0 ( ) a 29.5 ( ) a, b < 0.05 HBV DNA, log IU/ml (min-max) 2.3 ( ) 2.6 ( ) 2.5 ( ) 2.8 ( ) HBsAg, log IU/ml (min-max) 3.1 ( ) 3.3 ( ) 3.3 ( ) 3.0 ( ) 0.06 Genotye c B (%) 41 (51.3) 46 (33.6) d 52 (49.5) e 81 (64.8) < C (%) 39 (48.7) 91 (66.4) d 53 (50.5) e 44 (35.2) HBeAg heatitis B e antigen, HBsAg heatitis B surface antigen, HBV heatitis B virus a < 0.05 comared with the age of nulliaras and regnant women b < 0.05 comared with the age of uererant women, 7 12 months ostartum c The HBV genotye was not determined in 37 women because HBV DNA level was too low to be detected, including 23 nulliaras, 4 regnant women, 5 uererants 7 12 months ostartum and 5 uererants 2 5 years ostartum. Additionally, there were one woman infected with genotye D and another woman infected with genotye G. Since the numbers for genotyes D and G were too low to be meaningfully comared, we did not include these three women in the table d = 0.010, 0.012, and < comared with the frequencies of genotyes B and C in nulliara grou, uererant 7 12 months ostartum and uererant 2 5 years ostartum resectively e = comared with the frequencies of genotyes B and C in uererant 2 5 years ostartum. The differences between any other grous had no statistical significance (all > 0.05) ositive HBeAg was somewhat decreased (Table 1), which is in agreement with the findings reorted in the literature [24]. The ositivity of HBeAg indicates active viral relication. When the Abbott reagents were used, the median HBV DNA level in HBeAg-ositive women was 8.1 log IU/ml, much higher than 2.7 log IU/ml in HBeAg-negative women [25]. When Roche reagents were used, the average HBV DNA level in HBeAg-ositive women was 7.4 log coies/ml, much higher than 2.7 log coies/ml in HBeAg-negative ones [26]. In the resent study, serum HBV DNA level was measured by the reagents made in China, and the results showed that regnant women with ositive and negative HBeAg was 7.88 and 2.62 log IU/ml, resectively, with a difference of 5.26 log IU/ml. Additionally, the frequencies of high ( 6 log IU/ ml), moderate (3 < 6 log IU/ml), and low (< 3 log IU/ml) viral loads detected in the resent study (Tables 3 and 5) were also consistent with the reorted results assayed with Abbott and Roche reagents [25, 26]. These results suggest that the reagents for quantification of HBV DNA made in China are also reliable. Thus, the data in the resent study reflected the real HBV DNA levels in these women. It is reorted that different HBV genotyes may vary in some extent in the viral relication [23]. B and C are the main HBV genotyes in China, and it is generally considered that viral load is higher in atients infected with genotye C than that in those infected with genotye B [23]. Among HBeAg ositive women in this study, the frequencies of genotyes B and C were similar among regnant and non-regnant grous resectively (Table 2). In HBeAg negative women, the frequency of genotye C was the highest in regnant women (Table 4). However, the levels of HBV DNA and HBsAg in the regnant women remained to be comarable to the levels in three other non-regnant women (Tables 2 and 4). Therefore, the slight difference in the frequencies of genotyes in the regnant and non-regnant women should have minimal influence on the HBV DNA levels in these women. Generally, lacental trohoblast cells can secrete various cytokines and hormones during regnancy to maintain regnant women in an immunosuressive status by regulating major histocomatibility comlex (MHC) or Th1/Th2 balance. For examle, MHC-I and II molecules are not exressed on trohoblast cells, which are not recognized by maternal immune system. Meanwhile, the exression of human leukocyte antigen G can inhibit the activity of natural killer cells and T cells [27]. In addition, indoleamine 2,3-dioxygenase secreted by trohoblast cells can inhibit the roliferation of T cells by degrading trytohan [28]. The elevation of estrogen and rogesterone during regnancy may also be involved in the immunosuression in regnant women [29]. The Table 5 The frequency of high, moderate and low level of HBV DNA in 487 carrier women with negative HBeAg HBV DNA, log IU/ml Nulliara (n = 104) Pregnancy (n = 142) 7 12 months ostartum (n = 110) 2 5 years ostartum (n = 131) < 3 (%) 71 (68.3) 99 (69.7) 72 (65.5) 87 (66.4) < 6 (%) 31 (29.8) 41 (28.9) 37 (33.6) 43 (32.8) (%) 2 (1.9) 2 (1.4) 1 (0.9) 1 (0.8) HBeAg heatitis B e antigen, HBV heatitis B virus

6 Xu et al. BMC Pregnancy and Childbirth (2018) 18:292 Page 6 of 7 immunosuressive status during regnancy makes regnant women be redisosed to reactivation of latent viral infections, such as cytomegalovirus, olyomavirus, and human aillomavirus [12 17, 30, 31]. However, since the levels of HBV DNA in the circulation may reflect the relication of HBV in heatocytes, the findings that the levels of HBV DNA, HBsAg, and HBeAg in regnant women are each comarable with those in the non-regnant women in the resent study indicate that the immunosuression during regnancy does not result in the enhanced relication of HBV. The direct evidence to clarify whether regnancy has influence on the relication of HBV is to longitudinally observe the changes of viral loads and antigen levels before and during regnancy and ostartum in a same cohort of regnant women infected with HBV. However, the low feasibility of recruiting HBV-infected women just rior to concetion limited us to erform the longitudinal study. This is the main limitation of the resent study. Additionally, the serum samles were collected from the women who delivered their babies and from female atients who visited our hositals, which might be biased. However, we included a large number of HBV-infected women with similar roortion of HBeAg ositive women in each grou. The findings that regnant women did not show higher viral loads and viral antigen levels than the non-regnant women may at least rovide indirect evidence that regnancy has no influence on the relication of HBV. Pregnant women infected with HBV are more redisosed to have abnormal liver function tests than regnant women without HBV infection, but the liver function tests in the women included in the resent study were basically normal, which is the another limitation in this study. However, the observations that regnancy does not activate the relication of HBV suggest that liver injury in regnant women infected with HBV is less likely related to the reactivation of viral relication, which merits further study. Conclusion Based on the large numbers of articiating women infected with HBV in an endemic area, serum levels of HBV DNA and viral antigens in regnant women showed no significant changes comared with those in nulliarous and ostartum women. The results indicate that regnancy has minimal influence on the relication of HBV and the exression of viral antigens. Abbreviations anti-hbe: Antibody against HBeAg; HBeAg: Heatitis B e antigen; HBIG: Heatitis B immunoglobulin; HBsAg: Heatitis B surface antigen; HBV: Heatitis B virus; MHC: Histocomatibility comlex; PCR: Polymerase chain reaction Acknowledgements The authors would like to thank Ms. Wei Hu for her excellent technical suorts. Funding This work was suorted by the Jiangsu Provincial Deartment of Health (H and YXZXB ), the National Natural Science Foundation of China ( ), and the Science and Technology Deartment of Jiangsu Province (BK ), China. The funders had no role in study design, data collection and analysis, rearation and writing of the manuscrit and its submission. Availability of data and materials The data for this manuscrit are available from the corresonding author uon reasonable request. Authors contributions Conceived and designed the study: CX, JL, BX1, YH, and Y-HZ. Collected the clinical data: CX, LL, JC, BX2, and TC. Performed the laboratory tests: JL, YB, and JC. Performed the statistical analysis: BX1. Analyzed the data: CX, JL, LL, YB, BX1, JC, BX2, TC, YH, and Y-HZ. Wrote the manuscrit: CX and JL. Critically revised the manuscrit: Y-HZ. All authors have read and aroved the manuscrit. Ethics aroval and consent to articiate The study, including the informed consent form, was aroved by the institutional ethics review committee of Zhenjiang Fourth Peole s Hosital, Taixing Peole s Hosital, and Nanjing Drum Tower Hosital, resectively, and was erformed in accordance with the ethical standards laid down in the 1964 Declaration of Helsinki and its later amendments. Written informed consent was obtained from each articiant woman. Consent for ublication Not alicable. Cometing interests The authors declare that they have no cometing interests. Publisher s Note Sringer Nature remains neutral with regard to jurisdictional claims in ublished mas and institutional affiliations. Author details 1 Deartment of Obstetrics and Gynecology, Zhenjiang Fourth Peole s Hosital, Zhenjiang , Jiangsu, China. 2 Deartments of Laboratory Medicine and Infectious Diseases, Nanjing Drum Tower Hosital and Jiangsu Key Laboratory for Molecular Medicine, Nanjing University Medical School, 321 Zhongshan Road, Nanjing , Jiangsu, China. 3 Deartment of Obstetrics and Gynecology, Taixing Peole s Hosital, Taixing , Jiangsu, China. 4 Deartment of Biostatistics, Nanjing Drum Tower Hosital, Nanjing University Medical School, Nanjing , Jiangsu, China. 5 Deartment of Obstetrics and Gynecology, Nanjing Drum Tower Hosital, Nanjing University Medical School, Nanjing , Jiangsu, China. 6 Deartment of Infectious Diseases, Nanjing Drum Tower Hosital, Nanjing Medical University, Nanjing , Jiangsu, China. Received: 14 March 2018 Acceted: 2 July 2018 References 1. Schweitzer A, Horn J, Mikolajczyk RT, Krause G, Ott JJ. 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